Retinoblastoma, the most common intraocular tumor in children, is a prototypical model for studying tumor suppression. The identification of the first human cancer suppressor gene (retinoblastoma susceptibility gene) marked a new era of cancer research. Introducing the wild type RB gene into several RB deficient human tumor cells suppresses their neoplastic phenotypes including tumorigenic ability in nude mice. This finding substantiated the claim that RB is a tumor suppressor gene. Based on these important discoveries, we propose three interrelated projects to further explore the biochemical and cellular function of the retinoblastoma protein in order to fully understand how RB suppresses tumor formation. The major aims of these projects are as follows: I. To analyze the RB protein biochemically. RB proteins will be expressed in bacteria and purified to homogeneity for structural/functional domains mapping by partial proteolysis and for characterizing its polymerization property. II. To determine biochemical and biological significance of RB phosphorylation. By using site-directed mutagenesis the 12 potential cdc2 phosphorylation sites will be systematically altered either individually or in combination. Mutants will be tested in vitro for their ability to bind T antigen and other associated proteins or in vivo for their ability to suppress neoplastic phenotypes. III. To elucidate the RB protein interactive network by cloning and identifying its associated proteins (Aps). Two complementary cloning approaches either in vitro by direct screening expression libraries with RB protein as probe or in vivo using yeast GAL4 DNA binding and transcriptional activation domain system will be taken. Among dozens of clones isolated, five were chosen to be characterized first by sequence analysis of entire cDNAs, defining RB-binding domains of the Aps, generating antibodies against each Aps and exploring their roles in the progression of cell growth and differentiation. Results of these studies will contribute significantly to a basic understanding of retinoblastoma genesis as well as human oncogenesis in general.